When 3-hemolytic streptococci of Lancefield Group A are

Transcription

1 A LETHAL AGENT PRODUCED BY THE HEMOLYTIC STREPTOCOCCUS' T. N. HARRIS Department of Bacteriology, The School of Medicine, University of Pennsylvania, Philadelphia Received for publication August 28, 1941 When 3-hemolytic streptococci of Lancefield Group A are grown on meat-extract broth and the streptococci are then removed, the sterile filtrate will cause immediate death on intravenous injection into rabbits or mice. A characteristic train of symptoms precedes death. The fact that injection of streptococcal culture supernatant can cause immediate death has been previously reported in the literature (Todd, 1938). It has been described, however, as the result of the injection of the whole culture supernatant, and regarded as one of the physiological effects of the hemolysin therein. Thus, Todd, in discussing the immediately lethal effect of hemolytic filtrates from cultures of streptococci grown in meat-extract broth attributes the lethal effect to the hemolysin (1938), and gives lethal effects in terms of hemolytic doses (1939). There are many physiologically potent substances found in broth-filtrates of the hemolytic streptococcus, and general differences of opinion appear in the literature as to the identity of these factors. The present study was undertaken in order to investigate the relationship of the hemolysin and lethal agent, and to attempt to characterize the latter. METHODS Media Two media were used in this study, first a stock laboratory meat-infusion broth, and later one of simplified nature, almost protein-free. The first consisted of a filtered infusion of one pound of beef per liter, made up to 1 per cent of Difco neopeptone, 1 The expenses of this work have been defrayed by a grant from The Commonwealth Fund. 739

2 740 T. N. HARRIS 0.4 per cent anhydrous disodium phosphate, and 0.5 per cent glucose. Finally, sodium bicarbonate was added to a concentration of 0.1 molar. The simplified medium was based on 0.4 per cent Parke Davis peptone, which had been incubated for 90 minutes with 0.1 per cent trypsin (Difco 1:110). To this was added, per liter, 3.6 grams 1-asparagine, 1.8 grams monobasic potassium phosphate, 0.9 gram sodium citrate, and 20.0 ml. of yeast extract (Smythe, 1937). Sodium bicarbonate was added to a concentration of 0.1 molar and glucose to 0.5 per cent, in sterile solution. The medium as thus prepared gave no precipitate on saturation with ammonium sulfate. Strains The strains used were among those in use in this laboratory, 1685 and C203. No difference in total effect or symptoms could be observed between the filtrates of the two strains. Test-animals This work was done on white mice weighing 21 to 23 grams, from the ordinary laboratory stock, which had had no previous inoculation with streptococci or their products. The injections were made intravenously, employing the tail veins of the mouse. EXPERIMENTAL The Inoculation of Mice with Whole Culture Filtrate Symptoms. The end-point for lethal effect in mice was fairly sharp, and the trains of symptoms following, respectively, a lethal dose or a barely sublethal dose were quite characteristic. Immediately following the injection of a quantity of broth filtrate containing one lethal dose of this lethal agent (approximately 1 ml.), the following effects would be observed: The animal would lie on its side, extremely ill, with uncoordinated, sporadic movements of the limbs. There would be no generalized convulsions, except in response to mechanical stimuli. There would be no marked respiratory difficulty, and within a very few minutes there would be a final convulsive movement and then death.

3 LETHAL AGENT PRODUCED BY STREPTOCOCCUS A barely sub-lethal dose, about 10 per cent less than the lethal, would produce the following symptoms: The same ill appearance and sporadic movements would occur, with a generalized hyperacute trigger-like reflex response to mechanical stimulation or even to sharp noise. Frequently a sharp snap of the fingers would induce a generalized convulsion. There would be weakness of the muscles of the hind limbs, especially of the adductor groups, so that in few minutes, as the animal struggled to its feet, the hind legs would sprawl outward and locomotion would be poor. As the animal regained strength of its limbs the reflex hyper-irritability decreased progressively, until within about 15 minutes the animal would seem normal in every respect. If the dose was increased over the minimal lethal dose for that culture, death would occur after a shorter interval, until, if the dose was about 10 to 15 per cent greater than one MLD, the animal was usually dead on withdrawal of the injecting needle, that is within a few seconds. Sensitization, tolerance. No evidence was obtained of sensitization or tolerance to the lethal agent. In one group of 8 mice, injected almost daily with barely sub-lethal doses of a concentrate of broth filtrate, the MLD did not vary by more than its 10 per cent margin throughout the course of 8 injections, and the MLD three weeks later was still within that margin. Pathologic study. In view of the characteristic neurologic symptoms a pathologic examination of the brains of three mice was made. These animals had received very nearly sub-lethal doses 8 times in 11 days, and then were allowed to rest for two weeks before being sacrificed. Pathologic study of these brains showed anoxemic changes in the deep layers of the cortex and in the basal ganglia, with damage especially to the great ganglion cells. In one case there was a general irritation of the microglia throughout the cortex. There were also fresh hemorrhages and congestion in the basal region of the brain stem.2 Routes of iniection. The injection of several MLD intraperitoneally or intramuscularly produced no symptoms in mice. 2 I am indebted to Dr. F. H. Lewy of the University Hospital for the neuropathologic examinations.

4 742 T. N. HARRIS Other species. Although this work was done entirely on mice, two rabbits which were injected with larger amounts of broth concentrate also died immediately after injection. The neurologic symptoms were less clearly observed in these cases and could not be accurately described. MLD. For any one bottle of culture, the MLD was quite constant. The production of the lethal agent in different cultures showed a fairly constant optimum. The best titers obtained were 1.0 to 1.1 MLD per ml. of broth filtrate in 18-hour cultures, in both of the two media described. There was no significant increase of titer when the incubation was continued for another day. The production of this substance was, however, variable in different cultures, as was the production of other physiologically active substances in the streptococcal broth filtrate. Control injections. Many mice were injected with concentrates of sterile, uninoculated broth, as controls. It was found that the concentrate of 4 ml. or more of control broth, when injected intravenously, would cause death, but with entirely different symptoms. The animal would exhibit great respiratory distress, with labored heaving of the entire thorax, and die in a few minutes. No single constituent of the uninoculated broth could be identified as the cause of death. Concentrated solutions of the respective constituents were prepared, but in each case the amount contained in 12 to 20 ml. of broth was required in order to cause death. Differentiation of the Lethal Substance from Streptococcal Hemolysin Since the generally accepted view in the literature is that the lethal effect on injection is due to a hemolysin, experiments were done to determine whether the immediate death on injection was due to the hemolysin or to some other substance in the strepto. coccal filtrate. Accordingly, samples from a quantity of concentrated broth supernatant of known hemolytic titer and MLD were treated in the following ways, and the two titers determined before and after. 1. Reaction with anti-streptolysin. A portion of broth-concentrate was incubated for one hour at 370C. with rabbit antiserum prepared against whole streptococcal broth filtrate (Smythe

5 LETHAL AGENT PRODUCED BY STREPTOCOCCUS and Harris, 1940). The quantity of serum used containerd many times the anti-hemolysin required to neutralize the hemolysin present. After the'incubation the hemolytic titer was, of course, zero. The MLD was, however, unchanged, after correction had been made for the change in volume. 2. Cholesterol treatment. It has been found that the addition of a fine suspension of cholesterol crystals to a hemolytic filtrate of streptococcal cultures renders it non-hemolytic. In an attempt to distinguish the hemolysin from the lethal agent a portion of the concentrated broth-supernatant was incubated for 15 minutes at 370'C.'with a suspension of cholesterol (Nakayama, 1919). At the end of the incubation the' hemolytic titer was zero. The titer of the lethal effect was unchanged. 3. Destruction of hemolysin by heat. Another portion of the solution was placed in a water bath for 30 minutes. The fine precipitate which had formed was removed and the solution now had no'hemolytic effect, whereas the MLD had not changed. In these three procedures the hemolytic effect was destroyed without affecting measurably the lethal titer. Two other procedures were employed, however, to separate, chemically, hemolysin and'lethal agent while preserving both physiologic effects. 1. Dialysis. A portion of concentrated filtrate was dialyzed against- 20 volumes of distilled water, overnight. The next day the dialysate, on reconcentration in vacuo to the original volume of the bag, showed the same MLD but no hemolytic activity, whereas the dialyzed fluid was not lethal to mice and showed a hemolytic titer corresponding to the dilution incurred in dialysis. The symptoms were the same as described above. 2. Precipitation of hemolysin. Sodium chloride was added to a concentrated broth supernatant, almost to the point of precipitation of proteins. This solution was then placed in a water bath at 560C. for 15 minutes, and a fine white precipitate appeared. On separating the precipitate it was found that the supernatant had the same MLD as before, but a hemolytic titer of zero, whereas the precipitate, redissolved in tenth-molar sodium bicarbonate, showed no lethal effect, but retained a good part of the hemolytic effect. A final approach to this question was afforded by partial

6 '7AA T. K. K&DRIS purification of hemolysin. Using a chemical procedure previously described (1940), it ws -poible to employ a highly hemolytic solution, which had been obtained by concentrating the original broth with -respect to the hemolysin. A solution of this partially purified hemolysin was injected intravenously into mice, each animal receiving times the number of hemo- TABLE 1 The differentiation of lethal agent from hemolysin UxNs orl PROCEDURE SOLUTION TSTED HEMOLYTIC ACTIVTY MLD PEE ML. ml. I. Control for this experiment: M broth supernatant, concentrated 4X. II. Specific neutralization of Solution I + tenfold excess of hemolysin. antiserum for the hemolysin present III. Cholesterol inactivation Solution I + fivefold excess of of hemolysin. cholesterol suspension. IV. Destruction of hemolysin Solution I heated to 560C. for by heat. 30 minutes. V. Dialysis overnight in a. Solution I-dialyzed standing water. b. Dialysate VI. Addition of sodium chlo- a. Supernatant ride and heating to b. Precipitate C. for 15 min. Redissolving of ppt. VII. Purification of hemolysin. Hemolysin powder, 1685M, mgm. per ml. * In this table the volumes have been corrected for volume change incurred in the procedure. lytic units ordinarily associated with one MLD of broth supernatant. There was no immediate effect on the animals injected (table 1). Characterization of the Lethal Agent In order to determine the nature of this substance, a number of experiments were performed with the whole broth supernatant, and with broth concentrated in vacuo to about one-quarter its

7 LETHAL AGENT PRODUCED BY STREPTOCOCCUS 745 original volume. These experiments were done on both the meatinfusion broth supernatant, and on the broth supernatant from cultures in the essentially protein-free medium. 1. Dialysis. After dialysis into distilled water and reconcentration to the original volume, the broth supernatant showed no appreciable loss of lethal effect. 2. Chloroform gel. After repeated treatment with the chloroform gel, as described by Sevag, Lackman, and Smolens for removing proteins (1938), until no more material seemed to be removed from solution, it was found that the material removed by the gel had no lethal effect, and the supernatant had lost only a part of its lethal effect. S. Decolorization by charcoal. The broth supernatant concentrate was shaken with finely divided charcoal several times, until, after filtration, the solution was colorless. This colorless solution still showed the lethal effect in the same titer. 4. Heat stability. A quantity of broth supernatant concentrate was placed in a boiling water bath for 20 minutes. The flocculent precipitate was then removed by centrifugation. The supernatant when tested showed no diminution in lethal titer. 5. Acid and alkali-tolerance. The above experiment was repeated on another sample which had been brought to a ph of approximately 2. After being heated at that ph the supernatant was neutralized and tested. After correction for volume changes the titer was found to be unchanged. A similar experiment was done at a ph of approximately 11. At this ph there was some loss of lethal effect noted after treatment for an hour in the boiling water bath. 6. Effect of proteolytic enzyme. A sample of broth supernatant was incubated overnight at 370C. with trypsin (Difco 1:110) to a final concentration of 1 per cent of trypsin. There was no demonstrable change in lethal titer. 7. Alcohol precipitability. The lethal agent was precipitated from a concentrate of the broth supernatant of meat-infusion cultures by 5 volumes of alcohol, but not by 2 volumes. It could not, however, be precipitated from the protein-free medium by alcohol at all.

8 746 T. N. HARRIS 8. Cholesterol adsorption. Addition of stable cholesterol suspension did not neutralize the lethal effect, nor was the lethal substance removed by adsorption on crystals of cholesterol. 9. Lead acetate precipitability. The lethal substance was not precipitated from solution by lead acetate, as ordinarily prepared, nor by basic lead acetate (table 2). TABLE 2 Chemical characteristics of the lethal agent PROCEDURE MLD 1. Control solution: C203 broth supernatant, 3l times concentrated Dialysis (dialysate, reconcentrated to original volume of bag) Chloroform gel: a. Gel (dried, taken up in original volume of water). 1.5 b. Supernatant Decolorization by shaking with charcoal Heating in boiling water bath, 1 hour (5), at ph (5), at ph approximately Incubation with trypsin, 1 per cent, overnight at 37 C Incubation with equal volume of fine cholesterol suspension (0.05 mgm. per ml.) ml. DISCUSSION The data summarized above indicate that there is a substance produced in broth cultures of the hemolytic streptococcus, Group A, which causes immediate death in mice on intravenous injection, and characteristic neuromuscular effects in smaller doses. This substance would seem to be distinct from the soluble hemolysin produced in such cultures. All tests indicate it to be non-protein in nature, and it is probably a small, alcohol-soluble molecule, colorless in aqueous solution. The precipitation by sufficient alcohol from meat-infusion broth, and the precipitation of about 40 per cent from this broth by saturation with ammonium sulfate may be due to a non-specific inclusion in the mass of protein precipitate, because of the failure of precipitation by alcohol in solutions containing much less protein.

9 LETHAL AGENT PRODUCED BY STREPTOCOCCUS 747 The lethal agent described here is apparently not the same substance as the "lethal toxin," referred to by several workers in recent years (Nakayama, 1919, Abdalla and McLeod 1939). An entirely different lethal effect has been ascribed to culturesupernatants of the hemolytic streptococcus by these authors. In general this consists of death of the animals after some hours or days, usually within one day, following restlessness, anorexia, diarrhoea and other symptoms. These symptoms seem to begin after a latent period. Although there are the differences that one might expect among reports of various authors using grossly crude solutions of this other "lethal toxin" (the supernatant of whole culture), it would seem probable, on comparing the reports of Nakayama (1919), Pulvertaft (1929), Hartley (1928), Channon and McLeod (1929) and Abdalla and McLeod (1939), that all these authors are referring to the effects of the same substance. With respect to this substance also, there is difference of opinion as to the identity of the toxic substance with the hemolysin. Todd, however, (1938, 1939), may be reporting the effects of the lethal agent which is the subject of this paper, probably in combination with the effects of the "toxin" of these other authors, although he finds the lethal substance to be heat-labile. According to Todd the lethal effect he describes is due to an hemolysin. The work reported here is not consistent with that view. In general, it is dangerous to identify with certainty the products with which different authors have worked, and it must be done here with reservations. This is because of the lack of constant conditions of production, and especially because of the fact that almost all of the work reported has been done on whole broth supernatant. The multiplicity of physiologic effects produced by using whole broth supernatant hinders the identification of individual effects, although it is with one or two specific physiologic effects that each author has been concerned. It is probable that the clarification of the problem of lethal substances produced by the streptococcus, and their relations to each other and to the hemolysin, must await further characterization of each one, and separation of the substances from each other, insofar as that may be possible.

10 748 T. N. HARRIS SUMMARY A substance produced in broth in which hemolytic streptococci have grown causes immediate death on intravenous injection into mice. This substance is probably not the hemolysin. Points of difference from the hemolysin are presented, and some early steps in the chemical characterization of this substance are described. REFERENCES ABDALLA, N. W., AND MCLEOD, J. W The antigenic properties of streptolysin. Brit. J. Exptl. Path., 20, CHANNON, H. A., AND McLEOD, J. W On the importance of thermo-labile streptococcal toxin, with special reference to its cytolytic effect on leucocytes. J. Path. Bact., 32, HARTLEY, P Experiments on the purification and concentration of scarlet fever toxin. Brit. J. Exptl. Path., 9, HEWITT, L. F., AND TODD, E. W The effect of cholesterol and of sera contaminated with bacteria on the haemolysins produced by haemolytic streptococci. J. Path. Bact., 49, NAKAYAMA, Y Observations on streptolysin. J. Infectious Diseases, 25, PULVERTAFT, R. J. V An examination of the pathological effects of streptococcal toxin and haemolysin on rabbits, with special reference to the etiology of purpura fulminans. Lancet, 2, SEVAG, M. G., LACKMAN, D. B., AND SMOLENS, J The isolation of the components of streptococcal nucleoproteins in serologically active form. J. Biol. Chem., 124, SMYTHE, C. V An improved method of preparing hexose monophosphate from yeast extract. J. Biol. Chem., 118, SMYTHE, C. V., AND HARRIS, T. N Some properties of a hemolysin produced by Group A B-hemolytic streptococci. J. Immunol., 38, TODD, E. W Lethal toxins of haemolytic streptococci and their antibodies. Brit. J. Exptl. Path., 19,